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Concept: Aphthovirus

146

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these ‘look-a-like’ diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.

Concepts: Assay, Aphthovirus, Foot-and-mouth disease, Laboratory techniques, South America, Animal virology, Americas, Swine vesicular disease

141

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intra-typic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-coding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT 1, SAT 2, and SAT 3 serotypes. The seven serotype viruses were rescued successfully. Each chimeric FMDV with replacing P1 showed its serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Pigs vaccinated with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for customized vaccine with challenge tool to protect against epizootic disease from specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes significant economic losses. For the vaccine preparation, the selection of vaccine strains was complicated by its high antigenic variation. In the present study, we suggested that an effective strategy can be rapidly prepare and evaluate a mass-producing customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of seven representative serotype viruses. These chimeric viruses generally replicated readily in cell culture and had the similar particle size as the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses against all serotypes, especially for FMD-free countries which have prohibited import of FMDVs.

Concepts: Immune system, Microbiology, Virus, Vaccine, Vaccination, Influenza, Aphthovirus, Foot-and-mouth disease

141

Understanding the mechanisms of attenuation and virulence of foot-and-mouth disease virus (FMDV) in the natural host species is critical for development of next-generation countermeasures such as live-attenuated vaccines. Functional genomics analyses of FMDV have identified few virulence factors of which the leader proteinase (Lpro) is the most thoroughly investigated. Previous work from our laboratory has characterized host factors in cattle inoculated with virulent FMDV and attenuated mutant strains with transposon insertions within Lpro.

Concepts: Microbiology, Virology, Aphthovirus, Foot-and-mouth disease, Foot-and-mouth disease virus, Virulence, Virulence factor

139

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals. A synthetic vaccine candidate consisting of dendrimeric peptides harbouring two copies of a B-epitope [VP1(136-154)] linked to a T-cell epitope [3A(21-35)] of FMDV confers protection to type O FMDV challenge in pigs. Herein we show in cattle that novel dendrimeric peptides bearing a T-cell epitope [VP1(21-40] and two or four copies of a B-cell epitope [VP1(135-160)] from type O1 Campos FMDV (termed B2T and B4T, respectively) elicited FMDV specific immune responses to similar levels to a commercial vaccine. Animals were challenged with FMDV and 100% of vaccinated cattle with B2T or B4T were protected to podal generalization. Moreover, bovines immunized with B4T were completely protected (with no clinical signs) against FMDV challenge after three vaccine doses, which was associated with titers of viral neutralizing antibodies in serum higher than those of B2T group (p< 0.05) and levels of opsonic antibodies similar to those of animals immunized with one dose of FMDV commercial vaccine. Bovines vaccinated with both dendrimeric peptides presented high levels of IgG1 anti FMDV in sera and in mucosa. When IgA in nasal secretions was measured, 20% or 40% of the animals in B2T or B4T groups respectively, showed anti-FMDV IgA titers. In addition, B2T and B4T peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN-γ production assay. Taken together, these results support the potential of dendrimers B2T or B4T in cattle as a highly valuable, cost-effective FMDV candidate vaccine with DIVA potential.

Concepts: Immune system, Antibody, Infectious disease, Vaccination, Immunology, B cell, Aphthovirus, Foot-and-mouth disease

139

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output over wild-type 3C using a Gaussia luciferase reporter system. The novel point-mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline-arrays of FMDV virus-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.Importance: The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation provides a novel advancement for economical FMD vaccine production.

Concepts: DNA, Protein, Gene, Bacteria, Microbiology, Virology, Aphthovirus, Foot-and-mouth disease

139

Several foot-and-mouth disease virus (FMDV) carrier cattle were identified in Vietnam by the recovery of infectious virus from oropharyngeal fluid. This report contains the first near-complete genome sequences of seven viruses from sequential samples from one carrier animal collected over the course of 1 year. The characterization of within-host viral evolution has implications for FMDV control strategies.

Concepts: DNA, Gene, Organism, Virus, Infection, Aphthovirus, Foot-and-mouth disease, Foot-and-mouth disease virus

138

In 2015, foot-and-mouth disease (FMD) viruses of the A/ASIA/G-VII lineage emerged from the Indian sub-continent to cause outbreaks in the Middle and Near East. A factor which has been proposed to have contributed to the rapid spread of this lineage is the poor in vitro vaccine-match of field isolates to vaccine strains that are commonly used in the region. This study used data from outbreaks on four large-scale dairy farms using routine vaccination in Saudi Arabia, to evaluate the impact of vaccination and learn how to manage outbreaks more effectively in this setting. This evaluation also included an assessment of vaccine-induced neutralisation titres to the vaccine and field strains on a related farm with no history of FMD that employed an identical vaccination schedule. The incidence risk among exposed groups ranged from 2.6 to 20.1% and was significantly higher among youngstock (18.7%) compared to adults (7.4%). Evidence was found that local isolation of individual sick animals was more effective than whole group isolation and that subclinical infection and undetected circulation may occur on large-scale farms in Saudi Arabia, although both of these points require further evaluation. On the unaffected farm, the mean reciprocal titres for the vaccine and field strains were all above the cut-off supposed to correlate with clinical protection based on evidence from challenge studies. An estimate of vaccination effectiveness was not possible on the affected farms, but the incidence of FMD provides a more realistic estimation of the expected vaccine performance than in vivo studies or r1 value as it is based on field conditions and natural exposure. This study shows that analysis of field data from FMD outbreaks are a useful addition to more conventional challenge and in vitro based evaluations of vaccines and suggests further work is necessary to validate correlates of protection in field conditions.

Concepts: Vaccine, Vaccination, In vivo, Vaccination schedule, Saudi Arabia, Middle East, Aphthovirus, Foot-and-mouth disease

28

We designed a series of epitope proteins containing the G-H loops of three topotypes of foot-and-mouth disease virus (FMDV) serotype O and promiscuous artificial Th sites and selected one epitope protein (designated as B4) with optimal immunogenicity and cross-reactivity. Three out of five pigs immunized intramuscularly with this B4 were protected against virulent FMDV challenge after a single inoculation, while all pigs co-immunized with B4 and polyinosinic-cytidylic acid [poly (I: C)] conferred complete protection following FMDV challenge. Additionally, we demonstrated that all pigs co-immunized with B4 and poly (I: C) elicited FMDV-specific neutralizing antibodies, total IgG antibodies, typeIinterferon (IFN-α/β) and cytokines IFN-γ. In contrast, some pigs immunized with B4 alone produced parameters mentioned above, while some not, suggesting that poly (I: C) reduced animal-to-animal variations in both cellular and humoral responses often observed in association with epitope-based vaccines and up-regulated T-cell immunity often poorly observed in protein-based vaccines. We propose that poly (I: C) is an effective adjuvant for this epitope-based vaccine of FMDV. This combination could yield an effective and safe candidate vaccine for the control and eradication of FMD in pigs.

Concepts: Immune system, Antibody, Protein, Vaccination, Immunology, Smallpox, Aphthovirus, Foot-and-mouth disease

25

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Concepts: Immune system, Protein, Microbiology, Virus, Vaccine, Vaccination, Aphthovirus, Foot-and-mouth disease

24

Foot-and mouth disease virus is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype doesn’t confer inter-serotypic protection. However, some historical data have shown inter-serotypic protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study we have investigated the kinetics of the FMDV-specific antibody secreting cell response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar between all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O-, A- and Asia1-serotypes) a B cell response to FMDV SAT1 and C-serotype was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific antibody secreting cells (ASCs) in the absence of circulating FMDV-specific ASCs indicating the presence of short-lived ASCs; a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.Importance• Development of intra-serotypic response following a sequential vaccination regime of four different FMDV serotypes.• Indication of short-lived ASCs in the local lymphoid tissue - further evidence of a TI-2 type response to FMDV.

Concepts: Immune system, Antibody, Protein, Infectious disease, Virus, Aphthovirus, Foot-and-mouth disease, Foot-and-mouth disease virus